Biophysical evaluation of treating adipose tissue-derived stem cells using non-thermal atmospheric pressure plasma

Non-thermal atmospheric pressure plasma (NTAPP) is a partially ionized gas containing fast electrons and relatively slow ions. This study aims to investigate the influences of NTAPP on human adipose tissue-derived stem cells (ADSCs) and examine the feasibility of using optical spectroscopy as a non-destructive method for cell analysis. A plasma jet is used as the source of low-temperature plasma in which pure helium gas is ionized by a high voltage (8 kV) and frequency (6 kHz). ADSCs were exposed to the NTAPP for 30 s, 60 s, 90 s, and 120 s. The efficiency of the plasma treatment was investigated using flow cytometry and optical spectroscopy methods. This study compared surface markers of NTAPP treated and untreated ADSCs using CD90 and CD105 as positive markers. The result proved that NTAPP-exposed ADSCs maintain their stemming. Measuring ADSCS apoptosis by labeling Annexin V-Propidium Iodide showed that the plasma at short exposure time is relatively non-toxic. However, a longer exposure time can lead to apoptosis and necrosis. Moreover, Cell cycle analysis revealed that NTAPP accelerates the cell cycle in very low doses and can cause proliferation. In this experiment, flow cytometry measurements have been used to determine oxidative stress. The results showed that with increasing plasma dose, intracellular ROS levels reduced. This data also suggests that intracellular ROS are not responsible for the cells' viability. Furthermore, we used reflectance spectroscopy as a non-destructive method for evaluating treatment response and comparing this method with cell analysis techniques. The results indicate spectroscopy's efficiency as a method of cell analysis. This study suggests that NTAPP would be an efficient tool to improve ADSCs culture's efficiency in vitro; thus, we support the potential applications of NTAPP in the field of stem cell therapy and regenerative medicine.

Plasma generation and experimental setup. The plasma source used in this study was the plasma jet previously used in wound healing in diabetic rats 28 and treating diabetic foot ulcers 29 . The plasma jet used in this study consists of an insulating Pyrex tube (ID: 2 mm and OD: 4 mm) as a tube to control the discharge of working gas. A copper wire with 10 mm width was wrapped around the tube to work as a power electrode. The distance between the powered electrode and the nozzle tip was 10 mm. The virtual ground electrode of the space has been used in this configuration. Plasma was generated by applying a pulsed DC high voltage between the power electrode and the sample using a high voltage power supply. The repetition frequency, applied voltage, and duty ratio were 6 kHz, 8 kV, and 15%, respectively. A gas flow of 2 lit/min for 99.999% pure helium (He) was used. The distance between the nozzle tip and cells was 2 cm. The schematic of the experimental setup is shown in Fig. 1. Plasma treatment. Human ADSCs were isolated from adipose tissue and characterized as plastic adherent cells with fibroblastic morphology. After four successive passages, we obtained a homogeneous ADSCs population. Before treatment, 3 × 10 4 cells seeded in multiple well plates (6 well plates) were incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO2. Cells were exposed to the NTAPP for the 30 s, 60 s, 90 s, and 120 s. 0 s treatment cells were considered as the control group. The cells were incubated for 48 h after treatment. The distance between the cells and the device was fixed to 2 cm, and 1.5 ml of medium was used. Flow cytometry Analysis (Evaluation of ADSCs surface markers and Immunophenotype, Analysis of apoptosis, cell cycle analysis, and Detection of intracellular ROS) and Optical reflectance spectroscopy have been done 48 h after treatment. www.nature.com/scientificreports/ Evaluation of cell morphology after treatment. We examined and imaged morphological changes of the cells after plasma treatment using an IX70 inverted phase-contrast light microscope (Olympus, Japan).

Analysis of apoptosis. ADSCS apoptosis was measured through the labeling of Annexin V-Propidium
Iodide. This test is based on the protein Annexin V's ability to bind to phosphatidylserine (PS), which is translocated from the Interior cell membrane leaflet of viable cells to the outer in apoptotic cells (PS appears on the surface of necrotic cells as well). Propidium Iodide (PI) is a DNA-binding fluorescent reagent that penetrates damaged cell membranes. Adding PI allowed the distinguishing of viable cells (AnnVneg/PIneg), early apoptotic cells (AnnVpoz/PIneg), late apoptotic cells (AnnVpoz/PIpoz), and necrotic cells (AnnVneg/ PIpoz) 30,31 . The apoptosis assay was performed using Annexin V/ Pi method, following the manufacturer's instructions (Annexin V-FITC kit, Bender MedSystems, Austria). After treatment, the harvested cells were washed in PBS, resuspended in 100 μl of binding buffer, and stained with 5 μl FITC-conjugated Annexin-V for 15 min in the dark at room temperature. The samples were washed and resuspended in 250 μl binding buffer and then incubated with 5 μl Propidium Iodide (PI; Sigma-Aldrich, MO, USA) for 10 min. The results were analyzed on the BD FACSCalibur cytometer.
Cell cycle analysis. After treatment, the harvested cells were detached for the cell cycle analysis with 0.25% trypsin-EDTA (GIBCO, NY, USA), washed with PBS and then fixed with ice-cold 70% ethanol for 2 h. Cells were rewashed with PBS and treated with 50 μg/ml RNase A (Bio Basic Inc., ON, Canada) for 30 min. After that, cells were incubated with Propidium Iodide (PI; Sigma-Aldrich, MO, USA). Flow cytometry analysis was performed using the BD FACSCalibur cytometer.
Detection of intracellular ROS. 2, 7 -Dichlorofluorescin diacetate (DCFH-DA, Sigma), a stable nonfluorescent compound, was produced from dissolving 0.02 mmol of DCFH-DA in 1 mL of DMSO. DCFHDA underwent deacetylation by intracellular esterases when crossing the membrane and converted to DCFH. In the presence of active radicals within the cell, the non-fluorescent DCFH can oxidize and produce the highly Optical reflectance spectroscopy. We have used an optical reflectance spectroscopy system, a diffusion reflected one, as a non-destructive method for some physical analysis. Illumination for the spectra measurements was 400-1000 nm wavelength range. A miniature CCD-based fiber optic spectrometer (USB 2000; Ocean Optics) was applied (Fig. 7a). The spectroscopy experiments for recording reflectance spectra were conducted on the cell samples between the control group and the cell samples between 30, 60, 90, and 120 s of plasma treatment. To ensure more reliable cell spectroscopic signals and minimize bias in recording spectra, we first performed background subtraction of the plate and media (with or without soluble drug) as we had the option in the spectroscopy system 32-34 . Statistical and data analysis. Statistical analysis of data was performed using SPSS Statistics 21.0. Oneway analysis of variance (ANOVA) followed by Tukey's post-hoc test was performed to determine statistical significance. Data were considered statistically significant for P < 0.05 (marked as a single star), P < 0.01 (double star) and, P < 0.001 (triple star) were considered as extremely significant compared with the control. Flow cytometry data were acquired and analyzed using a FACSCalibur flow cytometer (BD Bioscience, CA, USA) as a prepaid service in out lab.

Results
Observation of morphological changes by microscopy. Normal ACSCs have a polyhedral shape and are adherent. However, dying cells shrunk, rounded and detached themselves from the plate. Our first observations indicate that with increasing plasma dose, the number of dying cells went up (Fig. 2). Our result shows that more and more cells shrunk, rounded, detached themselves from the plate, and finally died with increasing plasma dose. NTAPP-exposed ADSCs keep their stemming. To use NTAPP to enhance the proliferation of ADSCs for various applications, after exposure to NTAPP, the stem cell properties of ADSCs should be maintained. Characteristics of stemness of NTAPP treated and untreated ADSCs were compared using CD90 and CD105 as positive markers. The result demonstrated that both treated and untreated ADSCs showed high CD90 and CD105 expression (Fig. 3). These findings confirm that NTAPP treatment does not alter the characteristics of stemness of ADSCs.
Adipose tissue-derived stem cell death in response to non-thermal plasma. Staining of Annexin V / Propidium Iodide (AnnV / PI) is an assay based on the protein Annexin V being able to attach to the PS and PI ability to bind to DNA directly 35 . We can divide the data obtained from this test into four categories. Quadrant 4 (Live cells): Double negative as neither Annexin V nor PI staining could have been detected. Quadrant 3 (Early apoptotic cells): Annexin is positive, and PI is negative as the membrane stays intact. Quadrant 2 (Secondary necrotic and late apoptotic): Double-positive (membrane damage leads to pi staining penetration).
Quadrant 1 (Nuclei without plasma membrane): Necrotic cells with positive PI and negative Annexin (no membrane left for the Annexin to bind) 30,31 .
Plasma-treated ADSCs with short exposure times up to 30 s showed a few dead cells (mostly in late apoptotic and necrotic cell stages). However, with an increase in treatment time up to 60 s, fewer cells were destroyed (mostly in the apoptotic stage), verifying that the plasma at short exposures is relatively non-toxic. On the other hand, many dead cells were evident in the 90 s (Majorly in the necrotic cell stage) and 120 s (almost all in the double-positive stage) (Fig. 4A,C,D). We used the assay of the viability of cells with Propidium Iodide to confirm AnnV/PI results (Fig. 4B). Plasma-treated ADSCs with short exposure times showed a few dead cells; however, longer exposure times induce apoptosis and necrosis. In the control group, the largest proportion of cells belonged to phase G1 at around 63%. The figure for phase S stood at 22.46%, while that of phase G2 was lower at nearly 5% in the 0 s treatment group. In the 30 s and 60 s www.nature.com/scientificreports/ treatment, more cells belonged to phase S and G2 of the cell cycle than the control group. Nevertheless, fewer cells were in phase G1 in the 30 s and 60 s treatment than in the control group. Since proliferation increase would only be possible when the cell cycle is accelerated, these results suggest that NTAPP, in very low doses, caused a dose-dependent increase in the number of cells.
In the 90 s and 60 s treatment, we observed no significant difference in the proportion of cells in the phase S of the cell cycle compared to the control group (around 22%). More cells belonged to the G2 phase in these groups, whereas fewer cells existed in the G1 phase than in the control group. These results indicate a decrease in cell viability in a very high dose of plasma treatment. One of the main characteristics of apoptosis is the Fragmentation of the internucleosomal DNA. In the DNA frequency histograms, apoptotic cells can be categorized as cells with a fractional DNA content (sub-G1) 38 . In this experiment, increased cell death was observed with increasing plasma dose, and increased cell death was associated with a rise in the percentage of cells in the sub-G1 phase (Fig. 5a,b). NTAPP affects all cell cycle stages (Fig. 5c); however, the cell response is different depending on the cell cycle phase in which a cell exists.

Determination of intracellular ROS level after NTAPP treatment.
Suggesting that ROS contributes to regulating cellular functions of ADSCs, after NTAPP exposure, we evaluated the level of intracellular ROS in ADSCs. ROS continuously produced in normal cells at a restricted level. ROS level could be significantly increased in response to a wide range of pathophysiological. Extremely high ROS level will lead to biological macromolecules oxidization and cellular pathology, which finally leads to cell death 39,40 . Due to their short lifetimes, measuring ROS is highly challenging. In our study, flow cytometry measurements have been used to determine oxidative stress. The basis for this cellular assay is evaluating the fluorescence intensity [39][40][41] .
The flow cytometry measurements indicate the increase in ROS levels as a shift in the histograms showing the frequency distribution of DCF fluorescence values versus the number of events or cells. In this experiment, intracellular ROS levels were reduced with increasing plasma dose. The ROS level was lower in the 30 s and 60 s treatment than in cells in the untreated controls. Although the figure for the 30 s was approximately equal to that of the 60 s treatment, fewer cells were destroyed in the 60 s treatment than in the 30 s treatment (Fig. 6).
Optical reflectance spectroscopy. We used reflectance spectroscopy as a non-destructive method for evaluating treatment response and comparing this method with cell analysis techniques. There was a similarity in the refractive index of cell samples between 30 s and the control group. It is worth checking that there is a similarity in the stemness characteristics of ADSCs between the 30 s and 0 s treatment. Based on the reflectance spectra, in the 60 s treatment, the reflectance of samples decreased, and there was a peak around 800 nm, while in the control group and 60 s group, a similar trend was seen. There was a considerable decrease in the refractive index of cells in the 90 s treatment. In the group of 120 s irradiation, a very polished and reflected trend seems to be shown like a mirror, and it seems that the cellular characteristic is changing. Flow cytometry data seems to agree with the spectroscopic data, which evaluates the behavior of optical properties for control groups of 30 s, 60 s, and 90 s, showing the spectroscopy's efficiency as a method of cell analysis (Fig. 7). Reflectance spectros- www.nature.com/scientificreports/ copy was used as a non-destructive method for evaluating treatment response and comparing this method with cell analysis techniques. Our spectroscopic data shows the spectroscopy's efficiency as a cell analysis method.

Discussion
Up to now, NTAPP has been researched for its clinical application [42][43][44][45] ; however, several unanswered questions remain about the role of NTAPP in proliferation activation. It is worth mentioning that it is difficult to compare plasma sources thoroughly. The NTAPP working parameters are nearly impossible to standardize, and they are separately defined for every single source. This leads to a hinder in the interpretation of the plasma-induced effects. Therefore, similar experiments in many different laboratories using the same standardized source will be very useful to perform. The ADSCs isolated for this research shared the characteristics of other postnatal MSCs derived from various sources 46 , including typical fibroblastic morphology, adherence to plastic, and MSC specific surface markers expression. This study used a helium-based plasma jet as an NTAPP device generating multiple intracellular ROS/ RNS. To determine whether the NTAPP affected the viability of the ADSCs, primary cells isolated from adipose tissue were cultured and treated by plasma jet during four exposure times (30,60,90, and 120 s).
We demonstrated that NTAPP at short exposures is relatively non-toxic, although the number of dead cells rose as the exposure time increased. Cell cycle analysis suggests that NTAPP, in very low doses, promotes the proliferation of ADSCs while maintaining their stem cell surface markers. We also showed that, although there is a direct relationship between decreasing ROS and increasing cell death, increasing ROS level is not the only factor influencing cell death. Collectively, we strongly suggest that NTAPP can improve ADSCS culture's efficiency in vitro; thus, we support the potential applications of NTAPP in the field of stem cell therapy and regenerative medicine.
We may be able to destroy ADSCs without Considerable necrosis and subsequent inflammation by controlling plasma dose. We showed that prolonged exposure time to plasma could lead to cell death. In this study, the best time for cell proliferation was 60 s and the optimal time for cell death was 120 s, which did not cause necrosis. Our cell cycle data show that NTAPP affects all cell cycle stages; however, the cell response is different depending on the cell cycle phase in which a cell exists. Our results show that cells in the untreated group were mainly in phase G1. The short plasma exposure time led to an increase in the cell number in phase S and G2 and a decrease in cell number in phase G1 compared to the control group (No changes were observed in the phase sub G1). This suggests that NTAPP, in low doses, caused a dose-dependent increase in the number of cells and cell proliferation.
On the other hand, the long exposure time of plasma contributes to increased cell number in the G2 / M fraction and sub-G1 and decreases in cell number in phase G1. In phase S of the cell cycle, there was no significant difference in the number of cells in the 90 s and 120 s treatment compared to the control group. These results indicate increased cell death in a high dose of plasma treatment. Generally, plasma decreased the number of cells in phase G1, although the plasma effect on this phase is not dose-dependent. As we said, the control group cells were mostly in phase G1. With short-term plasma exposure, more cells were in phase S, and with a further increase in exposure time, most cells were in phase sub G1 compared to 0 s treatment.
Worth mentioning that although fewer dead cells were observed in the 60 s group than in the 30 s, the number of cells in phase S in the 30 s was higher. Generally, differentiation and proliferation are poorly compatible, and the process of differentiation is identified as a sequential event following weakened cell proliferation. In other words, when stem cells differentiate into specialized cells, intracellular signals and the growth factors responsible for cell growth are inhibited 47,48 . Therefore, we can suggest that the decrease in the number of cells in phase S in the 60 s treatment was possibly a prerequisite for their subsequent induction of differentiation. More research is required to describe these growth inhibitor properties and induce differentiation by NTAPP. www.nature.com/scientificreports/ As previously noted, it is not easy to fully describe plasma itself. Besides, its biologically active components are much more complicated, so it is hard to examine their effects, and the mechanisms involved are even more challenging to explain. Our initial step is that plasma generates a rich mixture of ROS and RNS, which are most likely to play a crucial role in the phenomena mentioned to date 13,49,50 . Historically, ROS and RNS have been considered 'bad' and strongly related to free-radical aging. However, nowadays, prevailing thought focuses on the role of ROS and RNS in a wide range of biological processes involved in the repair and protection of organisms and cells 51 . Although large doses of ROS and RNS are harmful to any cell and organism, the delivery of low dosage may help treat a wide variety of indications 12,13,[49][50][51][52] .
ROS could be in charge of the various biomedical effects of NTAPP [53][54][55] . At low concentrations, ROS can operate as a signaling molecule to modify cell processes and promote cell proliferation, differentiation, and migration [56][57][58] . ROS also helps stem cells maintain their stemness 59 . In contrast, higher ROS levels induce cell senescence and apoptosis, and too high concentrations of ROS cause non-specific cell death, which is probably necrosis [60][61][62] . Laurent et al. have indicated that low exogenous hydrogen peroxide level increased NIH 3T3 fibroblast proliferation while high concentrations of hydrogen peroxide have contributed to cell death 57 . In a study conducted by Dehui Xu (2015), it was shown that H2O2 and O2-are the main reactive species that trigger cell death. However, since each species alone was inadequate to obtain this result and cells express various iron proteins, they suggested that the OH radical produced by the Haber-Weiss reaction causes cell death. Iron proteins such as lactoferrin receptor, transferrin, and ferritin could catalyze H2O2 and O2-radicals into extremely reactive OH radicals that could seriously damage several macromolecules, such as DNA, lipids, and proteins 14 .
On the other side, RNS such as Nitric oxide is associated with different physiological functions 63 , such as the proliferation and differentiation of neural stem cells 64 . Besides, NO plays an intracellular messenger role in biological functions, including apoptosis [65][66][67] . A low NO level promotes cell proliferation 68 , and a high NO level contributes to cell cycle arrest and cell death 69 . In 2016, Jeongyeon Park and her team showed that helium-based NAPP increased the proliferation of ADSCs by activating ERK1/2, Akt, and their downstream NF-κ B via NO. Their result showed that the increased proliferation of ADSCs is triggered by NO rather than ROS 19,70,71 . Another study in 2015 showed that nitrogen species alone, such as electronically excited N2 and N ions, do not lead to cell death, and the production of RNS, such as NO or peroxynitrite (ONOO-) which can cause cell death, requires the presence of oxygen to be useful 12 . This emphasizes the significance of the presence of oxygen for plasma-induced cell death. In 2020, Park J et al. studied how CAP activates stem cell proliferation through epigenetic mechanisms. After analyzing the entire genome expression profiles of ASCs, they discovered that CAP upregulated genes for chemokines, cytokines, and growth factors while downregulating genes for intrinsic apoptotic pathways. They showed that NO produced from CAP was mainly responsible for plasma-induced epigenetic modifications at the mRNA and protein levels 71 .
Therefore, while some studies prove that ROS positively affects cell proliferation and some show they do not, all studies claim that ROS can cause cell death in higher doses. RNS, on the other hand, cannot cause cell death on their own, even in high doses, and the presence of oxygen is required for effective cell killing by plasma. However, RNS are the main component responsible for cell proliferation.
We also measured the intracellular ROS level in ADSCs after NTAPP exposure using DCFH-DA, a fluorogenic dye that monitored ROS activity. In our study, although cell death was different between the 30 s and 60 s of treatment, ROS level in 30 s treatment was the same as 60 s treatment. This data suggest that intracellular ROS are not responsible for the viability of cells. It is worth mentioning that intracellular ROS in a higher dose of plasma treatment decreased dose-dependent. This can be caused by a decrease in the total number of cells. Consistent with our result, other research groups have reported that the increased proliferation of ADSCs after NTAPP exposure is not triggered by intracellular ROS 70 .
We also used reflectance spectroscopy as a non-destructive way to evaluate treatment response and compare this approach to cell analysis techniques. The data for the 30 s group overlap control group results, and it is important to point out that there is an overlap between characteristics of stemness of ADSCs in the 30 s and 0 s treatment. There was a dose-dependent decrease in the refractive index of samples in the 60 s and 90 s treatment compared to the control group. The 120 s figure shows reflective and mirror behavior, and the cellular characteristic appears to be changing. These results show the spectroscopy's efficiency as a method of cell analysis. The findings of this study reinforce the ability of NTAPP to control the viability of stem cells without changing their stemness. The effect of NTAPP on other stem cells needs to be studied to improve NTAPP as a valuable method for biomedical application.